Contents

Introduction

These are methods to screen for and assay Ferulic-acid Esterase activity.

Plate Screen

Materials

Ethyl ferulate solution (100mg/ml in dimethylformamide).

Agar plates of media appropriate to your microorganism.

If screening natural strains some find it helpful to eliminate glucose from the media to drive FAE secretion.

This means that you will have to make this media yourself and can't buy a premix.

Water

Agar or Agarose (agarose is preferred)

Method

1. Grow colonies on agar plates of appropriate media until colonies reach a decent size.
2. For each plate to be screened, add 25-30mg agar to 6ml of water (i.e. if your screening 3 plates thats 80mg agar to 18ml water).
3. Microwave the agar mix until the agar is melted and put in 60°C water bath.
4. Once the media has been in the water bath for 10 mins:

1. Add the 20μL of ethyl ferulate solution for every ml of top agar (120μL per plate), and swirl lightly to disperse.

You want the ethyl ferulate to look cloudy in the agar so don't swirl too hard.

Bubbles = Enemy

2. Pour onto grown colonies immediately.

5. Incubate for ~4 hours.
6. If a clear halo forms around the colony in the top agar then it's positive for FAE!!!

Notes

Donaghy et al. (1998) added the ethyl ferulate solution directly to the media plates at a final concentration of 2mg/mL while Hassan and Pattat (2011) added it to the top agar at a stated concentration of 0.05mg/ml. We've found that the hassan and pattat concentration is way too low to make the agar cloudy but 1mg/ml can work well in a pinch. -- Mike

Agarose instead of agar is better too for top agar.

Nitrophenyl Ferulic Acid Assay

Materials

Protein desalting columns

HEPES

sodium azide

Dnase

4-nitrophenyl ferulic acid

Method

Make Protein buffer

100mM hepes

10μg/mL sodium Azide

5μL/mL Dnase

Concentrate cellular proteins from 1mL culture into 100μL buffer

Make Substrate buffer

2.5mM 4-nitrophenyl ferulic acid

0.5MKPO4

Add 20μL protein to 80μL substrate

Incubate for 30 mins at 37°C

Notes

Spectrophotometric Assay

This method quantifies the release of free ferulic-acid from ethyl-ferulate or methyl-ferulate. It is dependent on their absorbant divergence at 340nm.